ABSTRACT Heparin-induced thrombocytopenia (HIT) is thrombotic disorder caused by immune complexes that develops in settings where unfractionated heparin (UFH) remains the standard anticoagulant. For those with thrombotic complications, alternative anticoagulants do not mitigate symptoms, provide only partial protection against recurrent thrombosis and carry a risk of bleeding for which there is no antidote. HIT is over-diagnosed because current assays do not distinguish pathogenic from non-pathogenic anti-PF4 antibodies (Abs). There is need for better understanding the pathogenesis of HIT to develop better diagnostics and rational, disease-specific, non- anticoagulant management. We recently described the crystal structure of PF4 in complex with a heparin- based pentamer and a HIT-like murine (m) Ab (KKO). We showed that an anti-PF4 mAb (RTO) that prevents PF4 from forming tetramers and higher-ordered immune complexes, prevents platelet activation by HIT Abs in vitro, and thrombocytopenia and thrombosis caused by HIT Abs in vivo. Based on these structures, we developed Class I PF4 monomeric mutants that are unable to form tetramers, and thereby inhibit propagation of oligomeric PF4/UFH complexes that bind KKO and human HIT Abs, activate FcR?IIA receptors, inhibit expression of tissue factor and cause less inhibition of UFH/antithrombin III cofactor activity than native PF4. We also developed Class II PF4 mutants that form antigen-negative tetramers as an alternative approach. In this proposal, we will analyze these two classes of PF4 variants to better understand the pathogenesis of HIT and to develop novel diagnostic and potential therapeutic tools. Specific Aims (SA)#1. Efficacy, safety, mechanism of action and structure of Class I and II PF4 mutants. These studies will examine how PF4, heparin and HIT Abs oligomerize to form higher-ordered antigenic complexes. We will determine whether these PF4 variants block immune complex formation in vitro and in a murine model of HIT while generating a favorable hemostatic profile in this thrombotic disease. SA#2. Generation and characterization of Class I human and mouse PF4 mice. In vivo studies will be extended using CRISPR/Cas9 technology to develop mice expressing Class I monomeric PF4 variants and to examine their effects in a murine model of HIT. SA#3. Utility of Class I monomeric PF4 variants to distinguish pathogenic from non-pathogenic HIT Abs. Class I PF4 monomer columns will be used to separate pathogenic HIT Abs that bind tetrameric PF4/polyanion complexes from non-pathogenic Abs that bind monomeric PF4 and block binding of pathogenic Abs. We will test a novel hypothesis that HIT is more prevalent in patients lacking such blocking Abs. Together the proposed studies combine biophysical studies, novel murine models and a new hypothesis involving human samples that will provide a better understanding of immune complex formation in HIT, develop a disease- specific ELISA, and potentially offer a basis for a rational non-anticoagulant approach to complement contemporary anti-thrombotic therapy to mitigate this serious autoimmune thrombotic disorder.